The long range goal of this project is to study structure-function relationships in nitric oxide synthase (NOS). NOS is the enzyme responsible for the oxidation of arginine to nitric oxide (NO). In recent years, nitric oxide has been recognized as a major physiological messenger molecule involved in the nervous, immune, and cardiovascular systems. Owing to the potency and importance of NO as a regulatory molecule, NOS is a complex enzyme under stringent control. The enzyme consists of a heme domain, where the actual oxidation of arginine occurs and an FMN/FAD domain that serves to shuttle electrons from NADPH to the heme domain. Between the heme and flavin domains is a linker that binds another regulatory molecule, calmodulin. In addition to heme, FMN, and FAD, NOS contains yet another cofactor, tetrahydrobiopterin. Our lab has solved the crystal structure of the catalytic heme domain of all 3 mammalian NOS isoforms. This has opened the way for using structure-based approaches for developing isoform-selective selective inhibitors. A combination of crystallography, in vivo/in vitro testing, computational chemistry, and synthetic chemistry will be used to develop novel isoform-selective inhibitors. In addition, new technologies will be incorporated into the problem of NOS dynamics and electron transfer. In particular, is the use of high field NMR spectroscopy to study domain interactions in hoio-NOS and laser flash photolysis methods for studying electron transfer processes.